Nerve growth factor (NGF) is a target-derived neurotrophic factor involved in neuronal survival and differentiation. The receptor proteins, TrkA and p75NTR, that mediate NGF signaling are localized in caveolae-like membrane fractions (CLMs), as are many intermediates of NGF signaling, suggesting these domains are essential for NGF function. Cholesterol is necessary for the formation of CLMs and it appears that cellular cholesterol has a major role in NGF signaling. In support, mouse models of Niemann-Pick C (NPC) disease, which results from cholesterol trafficking, exhibit neurodegeneration and a loss of neurotrophic responsiveness. Additionally, studies on Alzheimer's disease (AD) indicate that proteins encoded by some genes linked to AD are involved in cholesterol trafficking. These data suggest the hypothesis that defects in cholesterol uptake and/or metabolism give rise to defects in NGF receptor signaling and trafficking, which in turn, contributes to neurodegeneration. To test this hypothesis, NGF receptor signaling will be studied in cholesterol depleted PC12 cells. The activation of the major NGF signaling pathways will be studied using immunoblot analysis, and the role of cholesterol in the trafficking of NGF receptors to the cell surface will be defined. This will be determined by biotinylation and isolation of cell surface proteins to determine whether cholesterol-depletion leads to a loss of cell-surface expression of NGF receptors or decreased delivery of receptors to the cell surface. Using immunohistochemistry and density fractionation of cellular lysates to isolate distinct membrane microdomains, differences in the subcellular localization of receptors in cholesterol-depleted and control cells will be defined. Radiolabeled NGF binding assays will be used to determine if cholesterol depletion affects NGF binding to its receptors and subsequent internalization of TrkA. Using the NPC mouse, neuronal cells will be isolated and tested for the defects in receptor trafficking and signaling that are defined in the PC12 cell experiments. Finally, the effects of cholesterol depletion on NGF-stimulated gene expression will be examined. Gene chip analysis will be used to determine differences in NGF-stimulated gene expression in cholesterol-depleted PC12 cells relative to control and in neuronal cells derived from the NPC mouse relative to cells derived from control mice.